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Application of mir-192 in regulation of proliferation and differentiation of sheep skeletal muscle satellite cells

A technology of mir-192 and satellite cells, applied in biochemical equipment and methods, DNA/RNA fragments, material inspection products, etc., can solve problems such as unclear effects

Active Publication Date: 2018-05-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although miR-192 has been found to be involved in carcinogenesis, its role in myogenic differentiation and proliferation of skeletal muscle satellite cells remains unclear

Method used

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  • Application of mir-192 in regulation of proliferation and differentiation of sheep skeletal muscle satellite cells
  • Application of mir-192 in regulation of proliferation and differentiation of sheep skeletal muscle satellite cells
  • Application of mir-192 in regulation of proliferation and differentiation of sheep skeletal muscle satellite cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Study on the regulation of miR-192 on the proliferation and differentiation of sheep skeletal muscle satellite cells

[0026] According to the previous high-throughput sequencing of sheep skeletal muscle, it was found that miR-192 (see SEQ ID NO: 1 in the sequence listing) was differentially expressed in sheep skeletal muscle during fetal and postnatal periods, and this result was verified by qPCR. Next, some basic biological characteristics of miR-192 were analyzed, including the position of miR-192 in the genome, the precursor of miR-192 (hairpin structure) and its position in the hairpin structure, miR-192 in Conservation among species. miR-192 is differentially expressed during the growth and development of sheep skeletal muscle, which may play a role in the growth and development of skeletal muscle.

[0027] The sheep skeletal muscle satellite cells were induced to differentiate, and the cells were collected at 0, 1, and 3 days of differentiation, and th...

Embodiment 2

[0034] Example 2 Detection of the Effect of miR-192 on the Expression of RB1 Protein in Sheep Skeletal Muscle Satellite Cells

[0035] miRNA is considered to regulate the expression of genes at the post-transcriptional level. Using Targetscan software to predict the target gene of miR-192, the 3' non-coding region of retinoblastoma gene 1 (Retinoblastoma1, RB1) has a target site of miR-192 (Such as Figure 5 shown), suggesting that RB1 may be a target gene of miR-192. RB1 is the first human tumor suppressor gene isolated and cloned. Because this gene is closely related to the occurrence of retinoblastoma, it is named as retinoblastoma gene. The RB1 gene has been shown to be involved in many growth and development regulation processes, including cell cycle regulation, cell senescence, apoptosis and growth inhibition.

[0036]Therefore, the present invention further detects whether miR-192 has an effect on the expression of RB1 protein level. First, according to the transfect...

Embodiment 3

[0047] Example 3 miR-192 inhibits RB1 gene expression by targeting the RB1 3' non-coding region

[0048] In order to verify whether miR-192 directly targets RB1, the present invention constructs a dual-luciferase vector containing a miR-192 target site and a dual-luciferase vector containing a mutated miR-192 target site. First, a nucleotide sequence containing the target site of miR-192 in the 3' non-coding region of RB1 was ligated into the dual-luciferase vector. The specific operation steps are as follows:

[0049] Amplify a nucleotide sequence containing the target site in the RB13' non-coding region from the sheep genome, the target site is as follows Figure 5 所示,以绵羊基因组DNA为模板扩增目的片段,其核苷酸序列如序列表中SEQ ID No:2所示,TTAACTTCAGCATGGTCTTACACTGGCACATTCAAAGCCACATTATTTCTAGGCCACAGTGCAGGTGATGAAAGGTCTTTGGGTTAGGCATTAATGTTTCTCTCTCTGATTTTGTGCAGAAGCTTCAAATTAAAATAGCTACAGTAGAAAAAGAGGCGCTTTCCCCCTCCCCCAACACCTAAAGGTGTAATTTAAACTATCTTGTGTGATTAACTTATTTAGAGATGGTATAATTTAAAATAGGTGGTATTTAAGGTAGGATCTACT...

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Abstract

The invention discloses the application of a non-coding small RNA miR-192 in inhibiting the myogenic differentiation of sheep skeletal muscle satellite cells and promoting the proliferation of sheep skeletal muscle satellite cells. The analog of the non-coding small RNA miR-192 is transfected into sheep The overexpression of intracellular miR‑192 in skeletal muscle satellite cells, compared with the cells overexpressing the control nucleotide sequence, the differentiation of sheep skeletal muscle satellite cells was inhibited, but the proliferation of sheep skeletal muscle satellite cells was inhibited. Capabilities are improved. The present invention also provides the application of miR-192 to inhibit the expression of RB1 protein level in sheep skeletal muscle satellite cells. The present invention discovers for the first time that miR-192 has the function of negatively regulating the myogenic differentiation of sheep skeletal muscle satellite cells and promoting the proliferation of sheep skeletal muscle satellite cells.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of miR-192 to regulate the proliferation and differentiation of sheep skeletal muscle satellite cells. Background technique [0002] Skeletal muscle satellite cells are a kind of muscle-derived stem cells, which are responsible for the regeneration of animal skeletal muscle after injury, and are an in vitro model for studying skeletal muscle growth and development. When skeletal muscle is injured, skeletal muscle satellite cells can quickly start the repair process, and can fuse into new myotubes in a short time, or fuse into damaged muscle fibers to repair muscle tissue. In developmental biology, the regeneration process after skeletal muscle injury is considered to be very similar to the development process of skeletal muscle, and skeletal muscle satellite cells are responsible for the regeneration of animal skeletal muscle after injury. Therefore, the myogenic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/6876C12Q1/02G01N33/68
Inventor 赵倩君马月辉赵谦关伟军浦亚斌康晔
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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